The urothelium is a stratified epithelium composed of Krt5-expressing basal cells, intermediate cells, and a luminal layer of umbrella cells that provide a crucial barrier between the urinary tract and blood. The urothelium contains stem cells that can regenerate the umbrella cell layer after acute damage, but chronic damage can compromise the urothelial barrier, leading to bladder dysfunction and persistent pain. Identification of urothelial stem cells and the signaling pathways that control them will be important for developing strategies for bladder augmentation and repair. Fate mapping studies reveal the existence of a Sonic hedgehog (Shh) expressing stem cell population in the adult urothelium that is proposed to be a basal cell. Our studies indicate that an Shh+ cell is also a stem cell in the embryonic urothelium, however direct analysis of the stem cell potential of basal cells using Krt5CreERT2;Rosa26 mice in fate mapping experiments reveals that they are unipotent in the adult and embryonic urothelium, suggesting that a different urothelial cell type i the stem cell. Consistent with this, we find that two other cell types are present in the Shh+ population, intermediate cells, and U-0 cells, a population that has not been previously described. We will test the stem cell potential intermediate and U-0 cells in Aims 1 and 2 in in the embryonic and adult regenerating urothelium, using CPP and uropathogenic E. coli as injury models, in collaboration with Indira Mysorekar's lab. Retinoic acid (RA) is a potent signaling molecule that can induce embryonic stem cells to differentiate into urothelial cells in culture, suggesting that retinoids may normally regulate urothelial progenitors. To address this, we used the ShhCre line to express a dominant inhibitory RA-receptor (RaraDN) which has been inserted into the Rosa26 locus, in the Shh+ population. We find that neither intermediate cells nor umbrella cells form in ShhCre/+;RaraDN mutants suggesting that retinoids are normally important in progenitor population for urothelial specification. We will directly address this question using cell type specific Cre lines to express RaraDN in intermediate and U-0 cells to determine which urothelial populations are mediators of RA-signaling during development and in the adult urothelium. These studies will identify a novel urothelial stem cell population, and will define the role of RA-signaling in formation and regeneration of the urothelium. The potential for RA to by used for induction of epithelial subtypes in the urothelium raises the possibility that a readily available and inexpensive compound could have novel therapeutic applications.